Virus-receptor interactions are often mediated by multifunctional viral attachment proteins that bind receptors and guide post-attachment cell-entry events. Key gaps in knowledge about these molecules include mechanisms by which receptor binding facilitates viral tropism and the conformational changes that orchestrate multiple activities in a single protein. The proposed research uses reovirus, a genetically tractable dsRNA virus that shows promise for oncolytic and vaccine applications, to dissect the process of specific and successful viral receptor engagement. Experiments will be performed to determine the role of glycan binding in viral tropism, define functions of viral attachment protein subsequent to receptor recognition, and elucidate mechanisms by which different capsid components attach to unique receptors. Following primary infection in the murine intestine, reovirus disseminates to the central nervous system (CNS), where it exhibits serotype-specific differences in tropism. Reovirus attachment is initiated by low-affinity binding to sialylated glycans followed by high-affinity binding to either junctional adhesion molecule-A (JAM-A) or Nogo receptor-1 (NgR1). Reovirus serotype 1 (T1) and serotype 3 (T3) strains bind to JAM-A and NgR1, but they vary in glycan utilization. The ?1 fiber protein binds glycan and JAM-A, whereas the ?3 capsid-surface protein binds NgR1. Three integrated specific aims are proposed to enhance knowledge of reovirus attachment mechanisms. In Specific Aim 1, the contribution of glycan engagement to reovirus neural tropism will be determined. Minimal carbohydrate-binding regions of T1 and T3 ?1 proteins will be defined using chimeric viruses engineered by reverse genetics. Mice will be infected with chimeric viruses to elucidate how glycan-binding specificity targets reovirus to discrete CNS sites. The specific glycan bound by reovirus on neurons will be identified. In Specific Aim 2, post-attachment functions and associated conformational changes in ?1 will be defined by testing neutralizing monoclonal antibodies (mAbs) specific for different ?1 conformations for the capacity to block viral attachment, internalization, and disassembly. Crystal structures of ?1 in complex with mAbs that impede distinct steps in viral entry will be determined to establish a biophysical basis for mAb-mediated infection blockade and identify new ?1 functional domains. Viral entry steps requiring ?1 conformational mobility will be defined using viruses with engineered disulfide bridges to lock ?1 in different conformational states. In Specific Aim 3, the structural basis of reovirus interactions with NgR1 will be elucidated. Sequences in ?3 and NgR1 required for binding and infection will be defined. The structure of NgR1 in complex with ?3 will be determined using X-ray crystallography and cryo-electron microscopy. These studies will enhance an understanding of mechanisms by which viruses engage cellular receptors, contribute new information about multifunctional viral attachment proteins, and accelerate the rational design of viral vectors for clinical applications.